Activated Partial Thromboplastin Time (aPTT)

Activated Partial Thromboplastin Time (aPTT) is a tested used to evaluate the intrinsic coagulation system. It aids the screen for congenital deficiencies of factors II, V, VIII, IX, X, XI and XII of the coagulation pathway. aPTT is also useful in the monitoring of heparin therapy. In addition, aPTT is used in the detection of Hemophilia A, Christmas disease, liver failure and Vitamin K deficiency. Furthermore, the test proveds screening for the presence of dysfibrinogenemia, disseminated intravascular coagulation, congenital deficiency of Fitzgerald factor and prekallikrein. aPTT is prolonged in the deficiency or inhition of any of the coagulation factors except factor VII. Coumadin therapy, heparin, aprotinin and lupus anticoagulants also have the same effect on aPTT. Prolonged aPTT is often evaluated with that of Prothrombin Time (PT) to assess coagulation system.


Coagulation Method

Coagulation Reaction Detection Method (Scattered Light Detection Method)

Irridates red light (660nm) onto a mixture of blood plasma and reagent and detects the change in turbidity (when fibrin clots are formed) as the change in scattered light. It measures the coagulation time.


Coagulation Point Detection Method (Percentage Detection Method)

Calculates the coagulation time as the time required to achieve the amount of scattered light that is set for the coagulation detection point, using the amount of scattered light that is present just after the start of detection as 0% and the amount of scattered light that is present at the completion of coagulation as 100%

Urine FEME

Urine FEME, also known as Urine Formed Elements with Microscopic Examination, consists of mainly two components. One component involves using reagent strips on patient's urine sample, while the other component involves microscopic examination of the urine.

The reagent strips contains nine separate reagent areas that contain different chemicals for the determination of a particular analyate. It also contains a non-reactive reagent pad which is used to determine the colour of the urine specimen. The automated machine used in the clinical laboratory is called the Clinitek Atlas analyzer. The machines contains the reagent strips mentioned above, and would be able to test several components in a urine specimen. The 12 components to be tested are pH, colour of urine, clarity, protein, occult blood, leukocytes, nitrite, glucose, ketone, bilirubin, urobilinogen and specific gravity. When a patient's urine sample reacts with the reagent pad, there would be a colour change on the respective reagent pad. A manual test can be carried out a spearate manual test reagent strip. The results can be read using a colour chart supplied by the manufacturer which indicate the presence or absence of some analytes, pH of urine or the level of analytes present in the urine specimen. These results may provide information regarding the patient's kidney function, urinary tract infections, carbohydrate metabolism and liver function

Firstly, 5ml of a patient's urine sample is poured into a clear plastic tube and labelled with the patient's particulars and barcode label. The tuve is then loaded onto a tube rack and loaded onto the analyzer. The machine would take up a small volume of the urine specimen and aliquot it onto the 10 reagent pads on the reagent strips. After which, the machine would detect the colour change on the test strip and the test results would be recorded.

The test results might give us some information of a patient's health status. For example, a presence of leukocytes could indicate a possible urinary tract infection. Presence of ketones could indicate a possible carbohydrate metabolic disorder or due to fasting. Presence of casts or blood in the urine could indicate a possible loss of kidney function.

The microscopic examination of the urine specimen involves charging a small volume of urine to a Kova microscopic slide. The slide is then examined using a light microscope and observed for the presence of RBC, WBC, epithelial cells, casts or crystals present in the urine. The results of microscopic examination should be cross-referenced to the results from the reagent strips, such as the presence of RBCs and WBCs. The results of the two different components should tally.

Streptococcus pneumoniae Antigen Test

Hi everyone. This is Jeremy (0702919B) from TG01 posting. This post will be about a test kit used in the lab to detect Streptococcus pneumoniae. Any questions, please feel free to ask.

Streptococcus pneumoniae Antigen Test

The BinaxNOW® Streptococcus pneumoniae Test is a rapid, in vitro immunochromatographic assay. It detects S. pneumoniae antigen in the urine of patients with pneumonia and also in the cerebral spinal fluid (CSF) of patients with meningitis. Both pneumonia and bacterial meningitis pose many complications to patients, and are almost always fatal if left untreated. Thus, it is necessary for an early detection and appropriate treatment to be administered. The Streptococcus pneumoniae Antigen Test, used together with culture and other methods, can also aid in the diagnosis of both pneumococcal pneumonia and pneumococcal meningitis.

Principle

The test kit involves the use of anti-S. pneumoniae antibody and control antibody. Both antibodies are adsorbed onto a nitrocellulose membrane (the anti-S. pneumoniae antibody on the sample line, while control antibody on the control line). The antibodies are conjugated to visualizing particles that are dried onto an inert fibrous support.

The presence of pneumococcal antigen in the sample will reacts to bind anti-S. pneumoniae conjugated antibody. The resulting antigen-conjugate complexes are captured by immobilized anti-S. pneumoniae antibody, forming the Sample Line. Immobilized control antibody captures anti-species conjugate, forming the Control Line.

Results Interpretation

Positive results:

Two pink-to-purple coloured lines will be formed. This means that an antigen was detected. Specimens with low levels of antigen may give a faint sample line. Any visible line is positive.

Negative results:

Only one pink-to-purple coloured control line in the top half of the window is formed. Formation of only the control line means that detection part of the test was done correctly, however no S. pneumoniae antigen was detected.

Invalid results:

No lines are seen, or only the sample line is seen. An invalid test must be repeated.

*Read results after 15 minutes.

That is all for the test kit. Thank you for reading.

G6PD screening & Quantitative assays

hello hello hello!
stella here for the final blog posting!
and i apologize for the late entry.

G6PD (Glucose-6-Phosphate Dehydrogenase) test is performed mainly on patients who develop symptoms of hemolytic anemia and neonatal jaundice. Hemolytic anemia takes place when the bone marrow is not able to compensate for the destruction by elevating the RBC production. G6PD is necessary for the maintenance of an intact red cell membrane because G6PD deficiency may lead to hemolysis of red cells. During the abnormal state, the RBCs will not be able to regenerate NADPH, a reaction that will be catalysed by G6PD in normal situations.

Both G6PD Quantitative and Qualitative (screening) assays are performed to enable differential diagnosis in G6PD deficiency to be made.
The principle of both G6PD assays is based on this reaction:

Glucose-6-Phosphate + NADP (no fluorescence) ---(G6PD)---> Gluconate-6-Phosphate + NADPH (fluorescence)

The NADPH produced will fluoresce under long-wave UV light during the reaction.
There is maerked deficiency or lack of enzyme G6PD when no fluorescnece is observed.
If Glucose-6-Phosphate is oxidised to Gluconate-6-Phosphate, the coenzyme NADP is reduced to NADPH with a corresponding elevation in fluorescence.

The procedures for both assays are different, with the screening assay much simpler as compared to the quantitative assay.

Steps for screening assay:
1) The EDTA consisting of patient's blood sample is inverted gently for several times for thorough mixing
2) 5ul of whole blood is added, 100ul of working reagent and 100ul of reagent blank into the wells of the titration plates
3) Working reagent is added and start timing
4) After 10 minutes, a small amount of reaction is taken and i spotted onto filter paper
5) The spots are dried for 5 mins using a hair-dryer
6) The spots are then viewed under long-wave UV light

Steps for quantitative assay:
1) The EDTA blood is washed 1 time with 0.9% saline
2) It is then sent for centrifugation to pack the cells at 3000rpm for 10 mins
3) The saline and the buffer coat are removed completely
4) 200ul of washed packed cells are pipetted into 200ul normal saline to obtain 1:1 rbc suspension
5) 50ul of this 1:1 suspension is pipetted into 250ul of !% saponin for lysis
6) It is then mixed well and left to stand for 10 mins at 2-8 degree celsius
7) The hemolysate is ready for testing


Reporting of results for Qualitative (screening) assay:
When fluorescence is observed, enzyme activity is indicated. This explains the presence of G6PD.


Reporting of results for Quantitative assay:
Reference ranges for adults/children = 7.2-17.4 ug/Hb
newborns/cord blood = 13.4-25.4 ug/Hb

Culturing Of Cord Blood

Hey!
This is Hakim (0703555C) doing my 4th and final blog posting.

Culturing of cord blood is essential for those who want to store their baby's cord blood.

Cord blood is the blood that is in the umbilical cord and placenta after the birth of the baby. The blood can be used to treat haematopoietic diseases such as leukemia, as the cord blood contains stem cells. The cord blood can be stored for up to 20 years for transfusion for future use to the baby or their family members. Therefore, it is essential that the cord blood is sterile and is free of any microorganisms. In addition, storage and maintenance of cord blood is very expensive.

The cord blood that is received for culturing is just a small amount of blood (2 eppendorf tubes).

The cord blood is cultured on 4 different agar plates:
1. TSA (Trypticase Soy Agar) with sheep blood stored in aerobic conditions - an enriched media to support a wide variety of microorganisms
2. TSA (Trypticase Soy Agar) with sheep blood stored in anaerobic conditions - an enriched media to support a wide variety of anaerobic microorganisms
3. MacConkey Agar- selective media for gram negative bacteria and also to differentiate between lactose fermenters and non-lactose fermenters
4. Sabouraud Dextrose Agar - selective media for yeast cells

The barcode number of the baby must be written on the underside of the media plate. A sterile swab is inserted into the cord blood and is innoculated onto the media plates. Then, a streaker is used to streak the plate.

The anaerobic TSA is inserted into an anaerobic jar. An anaerobic pack which absorbs oxygen and releases carbon dioxide is added into the jar before incubation. In addition, a control plate of a Mueller Hinton Agar innoculated with pseudomonas aeruginosa is put inside the anaerobic jar. The reason for this is because pseudomonas aeruginosa is a strict aerobe and it is the only organism that grows green colonies on Mueller Hinton. Therefore, if there isn't any green colonies on the agar after incubation, we can conclude that the conditions is an anaerobic condition.

After 24 hours of incubation, the agar plates are checked for any signs of bacterial growth. If there is no bacterial growth, the plates are incubated further for another 24 hours. If there is presence of bacterial growth, we must report the findings so that the cord blood will not be stored.

Please feel free to ask any questions!
Thanks!

Biochemical Tests

Good evening everyone. This is Jeremy (0702919B) from TG01. Today, I will be elaborating on several biochemical tests performed in the lab to aid in identifying bacteria.

Kliger’s Iron Agar (KIA)

KIA is a differential medium. It is similar to the Triple Sugar Iron (TSI) agar that we used in the school's lab. It tests for organisms’ abilities to ferment glucose and lactose to acid and acidic gas products. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose. These lactose non-fermenting enterics are generally more pathogenic in the gastrointestinal tract.

Glucose fermentation creates acidic byproducts that turn the phenol red indicator in the media yellow. Upon depletion of glucose, organisms that can ferment lactose will continue to produce acidic byproducts and the media will remain yellow. Gaseous products produce might lift the agar off the bottom of the tube.

Non lactose fermenters metabolize amino acids and proteins in the media, creating alkaline byproducts that turn the indicator red at the slant.

Non glucose and lactose fermenters will use solely amino acids and proteins, creating a red slant and unchanged color at the butt.

Organisms that sulfer reducing enteric will produce H₂S that creates a black precipitate at the butt.

	Slant	Butt	Examples
Lactose fermenters	Yellow	Yellow	Escherichia coli
Non lactose fermenters	Red	Yellow OR Black (H2S production)	Shigella dysenteriae Proteus mirabilis (produce black ppt)
Non glucose and lactose fermenters	Red	Unchanged	Pseudomonas aeruginosa

Simmons’ Citrate Agar

Simmons’ citrate agar tests the ability of an organism to use citrate as a sole carbon source and ammonium ions as a sole nitrogen source. The medium contains citrate, ammonium ions, and also a pH indicator, bromothymol blue. Organism that can grow on Simmons’ citrate agar are capable of metabolizing citrate as the sole carbon source and ammonium ions as the sole nitrogen source. This will create alkaline byproducts which will turn bromothymol blue indicator blue.

Growth on Simmon’s citrate agar or an intense blue color formation is an indication of a positive result.

Urease Test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea.

Well, that is all for now. Thank you for your attention.

Jeremy.

Immunohaematology (Blood Bank)

Firstly, I would like to discuss on how a patient's blood sample is being processed when it arrives at the blood bank. When a blood sample is first received, the Medical Technologist would check the EDTA blood tubes for any blood clots. This is being done by using 2-3 wooden sticks to "dig" the blood sample and try to "fish" out for any clots. If clots are absent, the sample would be suitable to be processed using the Ortho Autovue Innova System. If clots are present, the form accompanying the patient's sample should have "clotted" indicated on it. The blood sample with blood clots present cannot be processed by the Ortho Autovue Innova System, this the type and screen has to be carried manually.

After the blood samples in the EDTA are checked for presence of clots, the tube is loaded into the Ortho Autovue Innova System for the machine to read and screen the sample. The machine is able to carry out forward, reverse & Rh grouping of the blood samples. In addition, it is also able to detect abnormal antibodies that might be present in the blood. If the machine is malfunctioned, a manual type and screen can be carried out instead.

In type and screen which is done manually, forward group involves placing patient's blood sample into commercial sera. The 3 main groups of anti-sera are anti-A, anti-B & anti-AB. In reverse grouping, patient's serum (or plasma) is added to commercial blood. The 3 types of commericial blood used are blood group A, B & O. Lastly, to determine the presence of antigen D in patient's blood, patient's blood is added to anti-D commercial serum. This concludes the process of type and screen, and from this the patient's blood group and Rh status can be determined.

In an event that a patient requires blood transfusion, a cross-match must be carried out to check for compatiblity. In cross-matching, patient's serum is added into a sample of packet of blood (to be transfused). It is incubated at room temperature for 5 minutes, then centrifuged @ 3000rpm for 15s. The glass tube is inspected visually for any agglutination present. If there is no agglutination present, it can be concluded that the packet of blood can be safely transfused into the patient.

Regards,
Zhu Zhijie Dennis
0700847G
TG01

Oral administration of compound in animal models

Raymon

Oral administration

In a toxicity test involving oral administration of the drug/compound of interest, several methods can be considered mainly the oral gavaging and the dietary method
Gavaging is the process of force feeding whereby a tube is inserted into the mouth and down the oesophagus of the rat and the substance is subsequently emptied into the stomach via the syringe connected to the gavage tube.

Gavage VS Dietary method
The dietary method can be used if compound is suitable for mixing with the diet and that is stable under storage conditions similar to the diet. The compound must also be palatable to the animal.There are pros and cons to the dietary method. The advantage include beeing less laborious as compared to the need to gavage. The disadvantages include the exact concentration of compound for test which is mixed with food is hard to determine rather it is based on predicted food consumption and body weight. With the gavage method of dosing, a more precise amount of test compound can be delivered. However, there is a risk of punturing the oesophagus or even the lung if mishandled.

Gavage Techniques
1. Measure the distance from the tip of the nose to the last rib. This is the length of the needle that should be used. A curved needle is easiest to use and least likely to induce trauma
2. Fill syringe with appropriate amount of article to be dosed.
3. Restrain rat as mentioned in previous post
4. Place tip of the needle in rat's mouse
5. Slide ttip down back of the mouth, moving tip forward in one smooth motion
6. Needle should slide down the oesophagus easily. Resistance felt indicates that needle is not in place
7. Once everything is in place, administer the article

Fig 1: Gavaging of rat with tube

Fig 2: Measuring the tip and the anatomy of the rat

Fig 3: Gavage needles/tubes

Sexing and Gavaging of rats

Sex Determination

(extracted from http://research.uiowa.edu/animal/mp7.jpg)

Sexing is based upon anogenital distance. Male rats have a greater distance between the anus and urogenital opening. As compared to the females, males have a larger genital papilla. Well unlike the case of dogs, you cant tell the gender apart based on the testicles and phallus. Rats have open inguinal rings and can retract their phallus and testicles into their abdomen. How convenient. Thus the lack of testicles and phallus upon visual observation does not indicate that the rat is a female.

TDX analyzer

hello everybody!
can u believe it!
its going to be the 13th week of our SIP!
yay yay!

alright lets get things started.
this week i'm allocated to one of the analyzers being used in the lab called TDX analyzer.

This TDX analyzer perform tests for therapeutic drugs such as Cyclosporine, Everolimus and Methotrexate .
The therapeutic drugs mentioned are examples of immunosuppressive drugs.

The purpose of performing tests on the TDX analyzer is to quantitatively measure the amount of drug administered in the body to monitor the levels of these drugs to ensure therapeutic dosage and to minimise the levels of toxic side-effects,

im going to talk about one of the tests performed which is Cyclosporine test.
Cyclosporine is effective in fighting against tissue rejection after an organ transplantation.

The Cyclosporine Monoclonal Whole Blood assay uses FPIA (FLuorescence Polarization Immunoassay) Technology as the principle of the test.

This is when there will be a competitive protein binding technology that utilizes the specificity of immunoassay with a fluorescent tracer in order to differentiate the bound from the free analyte. The analyte, in this case, is a drug/drug derivative. During the process, the analyte is labelled. A specific Antibody against the analyte will then be prepared. The assay will cause the excitation of fluorescein tracer with polarized light and the level of fluoresent polarization of tracer is measured. The concentration of analyte is inversely proportional to the polarized light,

Methods for The Cyclosporine Monoclonal Whole Blood assay :
1. Microcentrifuge tubes are labelled accordingly for each of the patient's sample
2. The sample is mixed with gentle inversion and 150ul of it is pipetted out into the corresponding microcentrifuge tubes.
3. 300ul of Whole Blood Precipitation Reagent/ Probe Wash is pipetted accurately into each microcentrifuge tube.
4. Each tube is capped securely to avoid any spillage and vortexed for 10 seconds to make sure thorough mixing.
5. The specimens are centrifuged 10800rpm for 5 minutes to obtain a clear supernatant and a hard compact pellet of denatured proteins.
6. the supernatant is decant completely into the corresponding sample well of a sample cartridge (supernatant's minimum volume required is 150ul)
7. The Reagent Pack is mixed with gentle inversion. The formation of bubbles is avoided.
8. The reagent Pack is placed on the reagent platform.
9. The sample carousel is placed onto the analyzer.
10. Close the cover of the analyzer and press RUN.

Additional info must be noted that Cyclosporine must undergo a pretreatment step prior executing the methods above to extract the drug from the blood sample.

:)

Stella
0701059H

Influenza A & B Test Kits

Before I begin, I would like to apologize for the lateness of my entry, which was supposed to be posted a week ago.

At my workplace, I had the opportunity to use an Influenza A & B test kit to detect the presence of the virus which might be present in nasal swabs. We use a test kit to detect the presence of these two types of influenza. It should be noted that this is a qualitative and not quantitative test. If the patient shows a positive result, we would send out the test for PCR testing.

Above: The patient's nasal swab, the test kit and the extraction reagent solution vial.

Principles of test:
The test uses the principles of an immunochromatographic assay. As you can see from the picture above, the test kit has two sides to it: Testing for Influenza A, and testing for Influenza B. The picture actually shows a test kit that had already been done, which shows a negative for both A & B. The lines seen are the control lines.

If there is a positive result, another test line would appear nearer to the middle of the control line. The test area contains monoclonal anti-human influenza virus. If there is a presence of the virus in the specimen, the virus would bind to the antibody and react to form a purple-red line.

Methods of test:
A sterile swab should be inserted into the nasal cavity and rubbed on the mucosal surface for several times to collect mucous epidermis. The swab should be at a horizontal angle.
The swab is then dipped inside the extraction reagent solution vial, which contains buffer salt and detergent with 0.09% sodium azide.
The tip of the solution vial should be squeezed to contact with the swab and the swab should be rotated clockwise and anti-clockwise 5 times so that the virus (if any) would be transferred into the extraction reagent solution.
The swab is removed and the vial is gently shaken to mix the specimen.
The vial has a dropper on top. 4 drops of the solution is added to the specimen area and incubated at room temperature for 10 minutes.

We would have to write the results in a logbook for future reference. If the test shows negative for the influenzae, the swab as well as the test kit and the vial are disposed. If it's a positive we would have to put all 3 items in a ziplock bag and we would place it aside.

Hakim
0703555C

The Miscellaneous Bench

Hello everybody! This is Jeremy Chu (TG01) attached to bacteriology posting on...

The Miscellaneous Bench

The miscellaneous bench deals with every other specimens besides blood, stool, urine and respiratory specimens. Fluid, tissues and swabs are mainly the samples received. Some examples are heart/liver tissues, peritoneal fluid or peritoneum fluid, wound swabs and high/low vaginal swabs. Detecting bacteria at these sites are important, especially for sterile sites. There should not be any bacteria found at sterile sites such as the heart and liver or patient’s situation could be fatal.

Wound cultures are performed to isolate and identify bacteria infections causing an infection of the wound. This way, doctors can then administer the appropriate antibiotics to kill the causative agent.

Fluid culture is done, similarly to isolate and identify bacteria causing an infection. Peritoneal/Pericardial fluids are sterile fluids and any presence of bacteria is regarded as significant. Gram stain is also performed.

High vaginal swabs are cultured for the purpose of detecting group B streptococci (commonly Streptococcus agalactiae) found in the female reproductive tract. S. agalactiae can cause pneumonia and meningitis commonly in neonates and the elderly.

The different types or media used and its purposes are listed in the table below.

Media	Purpose	Types of specimens
Blood Agar	Enriched media to enhance bacteria growth	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices Vaginal swabs
MacConkey Agar	Selective media to inhibit most gram positive bacteria and differentiates lactose fermenters
Blood Agar (anaerobic)	Enriched media to enhance anaerobic bacteria growth	Tissues Fluids Fluid swabs
Cooked Meat broth	Enriched broth for cultivating bacteria	Tissues Fluids Fluid swabs Wound swabs Catheter tips/devices
LIM broth	Enriched broth used to grow group B streptococci from vaginal swabs	Vaginal swabs

Fluid culture agar plates are incubated under CO₂ conditions, while its broths in aerobic conditions. Blood, MAC, blood (anaerobic) agars and cooked meat broth are the choice of media.

Blood agar and LIM broth are used for vaginal swabs, while blood, MAC and cooked meat are used for ordinary wound cultures.

Roche Cobas e601 Analyzer

HI EVERYBODY!
stella here to post another entry!
I'm going to talk about Roche Cobas e601 Analyzer.

As i'm attached to a lab with mostly automated machines, therefore most of the tests are run on analyzers.

Roche Cobas e601 analyzer run different kinds of tests such as cardiac markers (CKMB, myoglobin, NTproBNP, Troponin T), tumour markers (AFP, CEA, CA125, CA15-3, free PSA, Total PSA) and bone markers (beta cross laps and osteocalcin)

An example of a test that is run on the Cobas e601 analyzer are cardiac markers such as Troponin T.
The purpose of performing the test is as follow:
Troponin T is useful and important in the diagnosis of myocardial damage such as myocardial infarction,

Principles of the tests : Immunoassay with Electrochemiluminescence technology

During the assay, the sample containing Troponin T is incubated with two monoclonal antibodies specific to the Troponin T. Examples of the antibodies against Troponin T are biotinylated monoclonalTroponin T-specific Antibody and a monoclonal troponin T-specific antibody labeled with a ruthenium complex. Thus, a sandwich complex is formed. Afterwhich, microparticles coated with streptavidin are added and the complex will be bound to to the solid phase through the interaction of biotin and streptavidin. The entire complex are drawn onto the electrode magnetically, with unbound components being washed away.
Electrochemiluminscent is induced by the application of a defined voltage. The photomultiplier measures the emission of light produced.

Reference interval for Troponin T:
<0.03ug/L

Troponin T can be futher divided into skeletal Troponin T and cardiac Troponin T.
Cardiac troponin T is a specific cardiac marker used for the diagnosis of myocardial damage.
It has unique cardiac specificity as it has different amino acid sequenes which are different from that of skeletal troponin T.
The presence of cardiac Troponin T in the bloodstream indicates there is myocardial damage.
In a healthy individual, no levels of troponin T can be detected in the bloodstream.
troponin T is a better cardiac marker due to its ability to stay in the bloodstream up to 2 weeks.
It rises from 4 to 6 hours, peaks at 12 to 24 hours and persist in the blood for 2 weeks.

Oral Glucose Tolerance Test

Hello everybody!

This is Hakim for another round of blogging.

NB: This is an edited entry. I have not removed anything, but have added in a few more points.


PURPOSE OF OGTT
The oral glucose tolerance test (OGTT) is a test performed to determine if the patient has diabetes. In my lab, majority of the patients who undergo this test is pregnant. This is because pregnant women could acquire gestational diabetes, which is a form of type II diabetes that is first diagnosed or begins during pregnancy. This condition usually ends soon after the person gives birth.

In a nutshell, the patient would have to give a urine sample and have their blood drawn out. After this, the patient is given a large dose of glucose and subsequently, urine is collected and blood is drawn after a period of time (depending on the GP's request). After the final urine collection and blood drawing, the urine samples are tested for glucose and ketones by using the dipstick. The blood tubes are centrifuged and its plasma is put in an analyzer to test for glucose levels.

PRINCIPLE OF TESTING PLASMA GLUCOSE LEVELS
Our lab is using the COBAS Integra 400 plus. It uses the principles of light absorbance and colorimetry. Glucose in the plasma is oxidised by glucose oxidase, producing gluconic acid and hydrogen peroxide. Peroxidase converts the hydrogen peroxide into oxygen and water. The oxygen liberated in the reaction is taken up by 4-aminophenazone with phenol and forms a pink-coloured substance. The substance can be measured at a wavelength of 515mm. A halogen lamp will produce a light beam and passes the cuvette. A photodetector would sense the amount of light passed through the cuvette (and also the amount of light absorbed by the substance). It is important that calibration is done frequently so that the results would be accurate.

PROCEDURE OF OGTT
It is important to note that the patient is required to fast for 8 hours before the test commences. This is done so as to prevent any pre-analytical variations. For example, a high plasma glucose after a meal could misdiagnose the patient as having impaired glucose tolerance (IGT). The patient is allowed to drink water during the fasting, but they are advised to try to drink as little as possible as the blood could be diluted if they take in large amounts of water. Also, the tube collected is a fluoride tube so that it will reduce the rate of glycolysis by the red blood cells so as to stabilize the levels of glucose. So after the first round of specimen collection, the patient is given a 75 gram glucose drink to drink. She has to finish the whole bottle. It tastes like concentrated F&N Orange. From my own experience, the drink is extremely sweet. I couldn't even finish the whole bottle (which is about 350mL) so I'm not sure which is more painful for the pregnant woman; childbirth or finishing up the whole bottle. There are 2 different timeframes for blood to be drawn, depending on the GP's request. It's gonna be quite confusing, so let's give an example. - Patient A is requested to have her blood and urine collected 1 hour AND 2 hours after drinking the glucose - Patient B is requested to have her blood and urine collected only 2 hours after drinking the glucose So after both patients finish the bottle, she comes back in either 1 hour (for patient A) or 2 hours time (for patient B), depending on the GP's request. During this time, she still cannot eat or drink anything (other than water, but in small amounts). Patient A comes back in 1 hour to have her blood and urine collected. At 2 hours after drinking the glucose, BOTH Patients A & B comes back to have their blood and urine collected. Only after this is done, they are able to eat and drink. For urine sample: The dipsticks are dipped into the urine to check for glucose and ketone levels. A high level of glucose in the urine would mean that there is a high level of glucose in the patient's bloodstream and the levels have exceeded the kidney's threshold. So the kidneys aren't able to reabsorb any more glucose and is then excreted through the urine. Ketone bodies are a result of lipid metabolism. This is caused by starvation. Ketone bodies would be present in diabetics. For blood sample: The blood tubes are centrifuged. The blood tubes MUST be labeled (0 hr, 1 hr, 2 hr) so as to prevent any confusion. The tubes are then put in an analyzer machine. For our lab, we are using the COBAS Integra 400 plus.

Note: For some reason, I can't upload pictures, so here is the link to the website that contains the picture of the analyzer.
http://labsystems.roche.com/content/products/integra_400plus/introduction.html
REFERENCE RANGE OF OGTT If the levels of plasma glucose is higher than normal, then we can say that the pregnant woman is suffering from gestational diabetes. There are many diagnosing criteria for OGTT. The World Health Organization's (WHO) guidelines for diagnosing GDM is ≥5.3 mmol/L for 0-hour plasma glucose, OR ≥10.0 mmol/L for 1-hour plasma glucose, OR ≥8.6 mmol/L for 2-hour plasma glucose. However, some doctors do not request for a 1-hour plasma glucose because it is not as clinically significant as the 0-hour and 2-hour plasma glucose levels.

They are advised to maintain a healthy lifestyle and have a proper suitable diet. They can also take insulin.

Ok that's all from me. If you have any questions for me, I'd be glad to answer them as fast as possible.

Happy SIP-ing!
Hakim
0703555C

13 more weeks!!!

Handling, restraining, sexing and identification of rats and mice

Hi all. I am currently working in the Animal Facility for a particular instiitution which cannot be named due to issue of confidentiality. I am juggling both my major project and my SIP thus it requires alot of initiative and time management. I usually assist in lab practicals and the maintenance of the animal husbandry while carrying out my experiment at the same time.

I shall share with you the Handling and restraining of the rat/mouse,the sexing and identification of the rats/mice which is essential as these techniques form the basics before other procedures can be carried out.

Whenever animals are used for research or teaching, it is important to minimise pain and distress that they may suffer as this would indirectly in turn affect the reliability and reproducibility of the experimental data. AThe adoption of the 3R principles as required by NACLAR Guiding Principles, such as refinement of procedures, replacement of animals and reduction of the number of animals used should become an integral part of all scientific research

HANDLING AND RESTRAINING TECHNIQUES
The use of proper handling and restraint techniques would help to minimiza unneccessary stress to both animals and research personnel.Its is recommended that the animal be handled on a regular basis in a non-threatening manner such as weighing, giving food treats etc. Most animals will lrespond positively to learn to recognize individuals. Suden movements prior to handling the animals may startle them.

Handling and restraint of Mice

1. Removal from cage
WHen handling mice, your movement should be slow and gentle.
Grasp the middle or near base of the tail between your thumb and your index finger, and lift the mouse out of the cage. Never hold it by the end of its tail. The mouse may also be picked up using a thumb forcep with its tips covered with rubber or polyethelene tubing to prevent damage to the mouse tail. Do not apply too much pressure when lifting the mouse as this may cause the tail to break.

2. Technical manipulation
Mouse has to be placed on a rough surface in which it can firmly hang on to such as the cage lid. Smooth surfaces will frighten the mouse because it cannot get a foothold. Gradual pressure can be applied to the tail while the index finger and thumb are used to scruff the skin around the animal's shoulder.ATurn the animal over to rest in the palm of your hand. Its tail may be restrained by the other hand or simply between the last 2 fingers of the hand holding the mice. THis enable the other hand to be free to carry out manipulations or injections.

Handling and restraint of Rats

1.Removal from cage and Manipulation
THe rat may be lifted out of the cage by the base of the tail. Never suspend the ray by the tail for too long. SUpport the body weight as soon as possible so as to minimise stress to it. Gently and firmly grasp the rat around the thorax with thumb and fore finger under each of the front legs. The rat might be stretched out by pulling the hind legs with the other hand.

Identification of Rat and Mice

THere are various ways for identification of rats and mice
1. Cage card - to identify strain of rat/mouse, sex, number, principal investigator and research protocols. This should not be removed from the cage to prevent misidentification
2. Pen marks on tail- this serves as a form of temporary identification of an individual. THe marks usually last for 1-2 days long
3. Hair clippings or dyeing the fur - may last up to 14 days
4. Ear tagging - small metal clips stamped with individual numbers applied near base of ear
5. Ear Punching and notching - rats can be identified with number 1 through 99 by piercing a hole or a notch or a double notch or any combination. 1,2 and 3 are holes punched on the right ear while holes punched on the left ear represents 10,20,30 (i dunno how to post pix)
while 4,5,6 and 40,50,60 are represented by notch and 7,8,9 and 70,80,90 are represented by double notches.

Major Project: Identification of compounds in tea that possess anti-microbial properties against Staphylococccus aureus

Hello, I'm Dennis from TG01 and I'm currently in school to complete my Major Project within 10 weeks before I move on to do my SIP, reason being the hospital that I'm attached to didn't have MPs to assign us with. Let me give you a brief overview of my MP.

The aim of my MP is to identify compounds present in tea that possess anti-microbial properties against Staphylococcus aureus. This is achieved by using a whole cell-based assay to test for anti-microbial activity of tea, followed by using High Performance Liquid Chromatography (HPLC) to identify specific compounds.

The cell based-assay in this case is used to test the anti-microbial properties of tea against S. aureus at different concentrations. The assay is made up of a 96-well micotitre plate, consisting of positive control, negative control and as well as different concentrations of tea. Ampicillin is as a standard.

My MP is actually a continuation on the work of a previous student doing her MP last year. She has completed the optimisation of the whole cell-based assay. What I had to do what to carry out some replicate experiments to ensure there was reproducibility of data. After I have ascertained the results I've obtained is reproducible, I can carry on to testing different types of tea for anti-microbial properties. There are several parts to my MP, so I'll be only covering the steps that are involved to determine the IC50 (Inhibitory Concentraion @ 50%) of Ampicillin

Firstly, I had to obtain pure colonies of S. aureus, and this is done by using the streak plate method on Tryptic Soy Agar (TSA). After overnight incubation of 18hours @ 37 degrees celcius, I would then subculture a pure colony of S. aureus 10ml of Tryptic Soy Broth (TSB). Again, it is incubated overnight for 18hours, at 37 degrees celcius and shaking at 150rpm.

Next, I would take 1ml of the culture and subculture it into 49ml of TSB (1:50 dilution). I would then allow the cells to grow to the log phase at the 3rd hour. The log phase has been determined by doing some experiments prior to this, whereby an absorbance curve is plotted using data obtained from the spectrophotometer.

After incubation of 3 hours, I would then use a hemocytometer to obtain the cell density of the culture. The culture was then diluted to obtain a final cell density of 1 X 10^6 cfu/ml. After the final cell density is obtained, the assay can then be set up. The assay consist of the following:

Postive Control: 40ul of cells + 10ul of Ampicillin (10ug/ml)
Negative Control: 40ul of cells + 10ul of autoclaved water
Data points: 40ul of cells + 10ul of Ampicillin (at various concentrations)

The whole microtitre plate incubate overnight at 37 degrees celcius. The results are read the next day using the luminescence method. This is somewhat similar to what we doing in the laboratory using the spectrophotometer. The difference is that the spectrophotometer measures turbity, while in this case, the luminescence is measure. A dye called resazurin is added into the individual wells. Resazurin is an oxidation-reduction indicator which changes from non-fluorescent blue to fluorescent pink by oxidoreductase within living bacteria. Simply put, if there is the presence of S. aureus in a particular well, resazurin would change from blue to pink. If there is absence of S. aureus in a particular well, resazurin remains blue.

The luminescence produced by resazurin is read using a machine which measures the amount of luminescence produced. The data is then plotted into a graph, and the IC50 of Ampicillin can be determined. The IC50 of Ampicillin is the concetration of ampicillin which inhibits 50% of S. aureus, and is useful when used to compare the IC50 of the different types of tea.

Please feel free to ask me questions, I'll do my best to answer them as soon as possible. Thank you.

Regards,
Zhu Zhijie Dennis
0700847G

DxC 800 analyzer-continued

Hello! this entry is an addition to explain my previous entry on the DxC analyzer. Some of you may not understand how the analyzers in the lab use e principles to carry out the different kinds of tests. So, I have added in one example of test used by the DxC 800 Beckman Coulter analyzer used in the lab I'm attached to :)

Clinical Chem Lab technique
Name of test: Renal Function Test on DxC 800 analyzer
Principle of test: ISE (Ion Selective Electrode) Method

The DxC 800 analyzer can determine Sodium ion concentration by indirect potentiometry using two glass Sodium electrodes, whereby one of them will be used as a reference electrode. A reference electrode is the electrode with stable and well-known electrode potential. The outer layer of the glass electrode must be hydrated adequately. The sodium concentration is measured using a precise volume of sample that is mixed with a buffered solution. When the sample buffer mixture gets in contact with the electrode, sodium ions in the sample will undergo an ion exchange process with the sodium ions in the hydrated layer of the electrode. Thus, there will be a change in electrode potential which will be referenced with the reference electrode. From here, the concentration of Sodium can be measured.

Normal reference range of test results are:
0 day to 12 years old: 131-144mmol/L
> 12 years old: 135-145 mmol/L

Any results higher or lower than the normal reference ranges indicate abnormalities in patients.

This sodium measurements is used in the diagnosis and treatment of renal failure, hypernatraemia, hyponatraemia as well as other diseases involving electrolyte imbalance.

I hope this helps you guys to understand better! :)

Stella
0701059H
TG01

The Urine Bench

Good evening lecturers and fellow course mates. My name is Chu De Ming Jeremy (admin no. 0702919B) and I am attached to Microbiology (Bacteriology) for the whole course of attachment. I will be sharing my experiences over the past 2 weeks in my assigned laboratory. I shall try to keep my posts as clean and concise as possible to ease the monotony.

Before I begin, I apologize for this late post and to make up for this, I will try to share my encounters weekly. Today, I shall start of with my very first week (22nd June - 27th June) in the lab. I title it, The Urine Bench.

The Urine Bench

Urine culture is a very common laboratory test performed to diagnose urinary tract infections (UTIs) which are frequently due to bacterial infections. Escherichia coli, Klebsiella, Enterobacter, Proteus and Pseudomonas sp. are the common culprits of UTIs.
Urine culture requires midstream urine as the specimen because the first bit of urine that passes out may be contaminated from the skin. This is to eliminate possible normal flora growth which can interfere in our identification of pathogenic microorganisms.

In the lab, I was tasked to perform urine cultures for the whole week. Specimens arrived in batches, either in the form of urine, fluid from the kidneys, or dipslides which has already been dipped in urine.

Urine specimen is used to perform aerobic cultures on a split blood agar plate and cysteine lactose-electrolyte-deficient (CLED) media. Direct plating is performed using a disposable 1 micron wired loop and the plates are later sent for incubation at 35°C overnight.
Percutaneous nephrostomy (PCN) is performed on fluid from kidneys. Procedures are similar to aerobic cultures of urine. The only difference is direct plating require the use of both 1 and 10 micron wired loop on 2 separate split blood/CLED agar plates.



A self drawn picture illustrating how streaking is done. Urine is streaked onto blood agar first, then onto the CLED agar. This is to allow trace back if there is any errors or contaminants encountered.




When dipslides are received, they are sent for incubation at 35°C overnight.










Picture of dipslides from http://www.solarbiologicals.com/pages/medical/images/cled-mac-2.JPG
One side is CLED media, the other can be MacConkey (MAC) agar or blood agar.

Blood agar is an enriched differential media that enhances the growth of fastidious microorganism.
CLED media, due to its electrolyte deficient property, prevents Proteus sp. from swarming. It also contains lactose which differentiates lactose fermenters (yellow colonies) from non-lactose fermenters (blue colonies).
MAC agar is a selective differential media that inhibit the growth of most Gram-positive bacteria to stain Gram-negative bacteria for lactose fermentation.

Dipslides are unpopular compared to plating on agar plates as their area is small, making it difficult to make observations. However, one advantage of dipslides is that it can be retained for 2 days before sending it for incubation, as compared to urine where it has to arrive fresh (not more than 24h if kept in refrigerator).

Well, that is all you will need to know for The Urine Bench! Any questions please do not feel hesitant to ask. Thank you.
Check back for the next post in the next couple of days.

Jeremy.